Epitopes formed by non-covalent association of conjugates

ABSTRACT

A method for producing and using in treatment a composition for interacting with a ligand, which composition comprises a non-covalent association of a plurality of distinct conjugates, each conjugate comprising a head group and a tail group, wherein the tail groups of the conjugates form a hydrophobic aggregation and the conjugates are movable within the association so that, in the presence of a ligand, at least two of the head groups are appropriately positioned to form an epitope capable of interacting with the ligand more strongly than each of the head groups individually.

The present invention relates to a composition for interacting with aligand, a method for producing such a composition and a method forproducing a molecule based on the composition.

BACKGROUND OF THE INVENTION

Protein receptors are known normally to bind to their target ligands viaepitopes, which constitute a small proportion of the total proteinmolecule. For maximum binding or interaction, the structure of theepitope needs to be maintained in a rigid conformation in order to forma binding site containing all the necessary components of the epitope inclose proximity. Attempts to produce an analogous peptide constructedsolely of the amino acids comprising the binding site often fail becausethese peptides do not possess the same biological activity as theprotein receptor. This is attributed to the peptide having a differentconformation in free solution from that of the entire protein receptor.In addition, where the binding site of a protein is constructed ofoligo-peptides from different, non-contiguous parts of a protein chain,mixing isolated oligopeptides in free solution does not result inreconstitution of the active binding site.

Being constrained to use such large proteins to present binding-siteepitopes gives rise to several problems in development of newreceptor-specific therapeutic strategies. One problem is that such largeproteins can readily evoke an immune response. A second problem is thatlong peptide chains are susceptible to attack by endopeptidases, such asthose in the lumen of the gut. Finally, these large proteins can becostly to manufacture, purify and maintain in stable form.

SUMMARY OF THE INVENTION

The present invention aims to overcome the disadvantages of the priorart.

In a first aspect, the invention provides a composition for interactingwith a ligand, which composition comprises a non-covalent assembly of aplurality of distinct conjugates, each conjugate comprising a head groupand a tail group, wherein the tail groups of the conjugates form ahydrophobic aggregation and the conjugates have freedom of motion withrespect to each other within the assembly so that, in the presence of aligand, at least two of the head groups (which are the same ordifferent) are appropriately positioned to form an epitope capable ofinteracting with the ligand more strongly than each of head groupsindividually. The head groups are typically hydrophilic and the tailgroups typically hydrophobic, eg lipophilic, composed of hydrocarbonchains, halophilic, constructed of fluorocarbon chains, or silane based.

By constructing conjugates with a head group and a tail group inaccordance with the present invention, the tail groups can associate toform a hydrophobic aggregation which is typically a supramolecularassembly such as a micelle, a lamellar structure, a liposome or otherlipid structure, in which the conjugate are oriented whereby the headgroups are brought into close proximity when in an aqueous phase.Because the conjugates are movable within the assembly, the head groupsare able to adopt a number of different positions within the assembly.The head groups, which are typically non-identical, are therefore freeto move within the assembly and, surprisingly, to interact cooperativelyto induce biological consequences which the head groups on their own arenot capable of eliciting. A further unexpected finding is thatassemblies composed of combinations of different headgroups are capableof eliciting biological responses or participating in binding withbiological receptors while assemblies composed of single headgroups arenot capable of acting in this way.

As indicated above, these supra-molecular assemblies are typicallyparticulate or colloidal in nature, usually comprising many hundreds ofsub-units (the conjugates) all oriented with the headgroups directedoutwards from the centre of the particle as shown in FIG. 1 a. Each ofthe conjugates may change its location within the assembly, being freeto exchange places with adjacent conjugates by a process of Brownianmotion and, in so doing, may migrate over the whole surface of theassembly. Other manifestations of supra-molecular assemblies are cubicphases and coated surfaces.

Each conjugate in the assembly may have a head group selected from onechemical or biological class or a number of different classes, such asan amino acid or peptide; a peptide analogue; a mono-, di- orpoly-saccharide; a mono-, di- or poly-nucleotide; a sterol; an alkaloid;an isoprenoid; an inositol derivative; a single or fused aromaticnucleus; a water-soluble vitamin; a porphyrin or haem nucleus; aphthalocyanine; a metal ion chelate; a water-soluble drug; a hormone; oran enzyme substrate.

In one preferred embodiment, each head group comprises an amino acid oroligo-peptide, which may be the terminal portion of a peptide chain. Itis desirable to keep the length of the peptide to a minimum so as toavoid eliciting an immune response where the composition is to be usedin vivo. Accordingly, it is preferred that the peptide is no more thansix amino acids long.

The amino acids employed can be any of the natural amino acids,substituted derivatives, analogues, and D-forms thereof.

The tail groups of the conjugates may be all the same or may be amixture of different tail groups, each of which preferably comprises ahydrophobic group selected from a linear, branched, cyclic, polycyclic,saturated or unsaturated construct, with or without hetero-atomsincluded in the structure which can be substituted or unsubstituted, forexample, a lipidic amino acid analogue; a prostaglandin; a leukotriene;a mono- or diglyceride; a sterol; a sphingosine or ceramide derivative;and a silicon or halogen-substituted derivative of such a hydrophobicgroup. The tail group preferably has from 6 to 24 carbon atoms and morepreferably comprises from 10 to 14 carbon atoms. More than one tailgroup may be present in each conjugate. For example, one or more lipidicamino acids with hydrocarbon side chains may form part of eachconjugate, linked to one or more amino acids in the head group.

Any chemical method may be used to link the head group to the tailgroup. For example, each conjugate may further comprise a spacer grouplinking the head group to the tail group so as to facilitatepresentation of the head group on the surface of the non-covalentassociation. Such spacer groups are well known and include, for example,amino acids, hydroxy acids, sugars and polyethylene glycol.

In a further aspect, the present invention provides a composition asdefined above, for use as a medicament, a prophylactic or a diagnostic.

An advantage of the invention is that strong specific bindinginteractions can be achieved with conjugates in which the head groupsare small in comparison to conventional biological receptors. If thehead group is an oligo-peptide, for example, then the length of thepeptide chain would not normally exceed ten amino acids and wouldpreferably be six or less. Accordingly, compositions according to thepresent invention can be made far less immunogenic than their proteincounterparts.

In accordance with this aspect of the invention, not only can thecomposition of the present invention be formulated to interact with aligand in vitro but also the composition can be used in vivo, optionallyformulated with a suitable diluent, excipient or carrier in accordancewith a suitable delivery route.

In a further aspect, the present invention provides use of a conjugatecomprising a head group and tail group for the preparation of thecomposition as defined above.

There is further provided a method for producing a composition forinteracting with a ligand, which method comprises:

-   (a) providing a plurality of distinct conjugates, each conjugate    comprising a head group and a tail group; and (b) forming from the    plurality of conjugates, by noncovalent association thereof, an    assembly in which the tail groups aggregate hydrophobically and in    which the conjugates exhibit freedom of motion relative to one    another so that, in the presence of a ligand, at least two of the    head groups are appropriately positioned to form an epitope capable    of interacting with the ligand more strongly than each of head    groups individually. Each conjugate is preferably as defined above.

The conjugates may be dispersed in aqueous phase by a variety of knownmethodologies for the preparation of lipid vesicles, includingmechanical mixing, exposure to high shear forces, sonication, solventdispersion or codissolution with detergents. Typically, the non-covalentsupra-molecular assemblies formed thereby will be composed of severaldifferent conjugates mixed together. Additional lipidic materials mayoptionally be added to alter surface properties, to aid in thedispersion of the conjugates, to stabilise the non-covalently associatedassembly of conjugates, to aid in the presentation of head groups of theconjugates, or to permit the construction of vehicles which can betargeted by the epitopes formed upon random movement of the conjugatesand appropriate positioning of the head groups within the assembly.

An important aspect of the method according to the present inventioninvolves the step of identifying the plurality of conjugates which hasthe desired biological activity. In a preferred aspect, this stepcomprises

-   (i) selecting a set of conjugates with an array of head groups;-   (ii) forming a non-covalent association therefrom, in which the tail    groups aggregate hydrophobically and in which the conjugates exhibit    freedom of motion with respect to one another;-   (iii) assaying for sufficient interaction between the non-covalent    association and the ligand;-   (iv) optionally repeating steps (i) to (iii) using a set of    conjugates with a modified array of head groups; and-   (v) on finding sufficient interaction in step (iii), selecting the    set of conjugates as the plurality of conjugates in step (a).

Examples of assays for “sufficient interaction” may include bindingassays such as those utilising the ELISA principle for detection ofassociation between antibody and antigen. Other suitable in vitro assaysinclude modification of fluorescence of environmentally-sensitivemembrane-bound fluorescent probes, precipitation reactions, enhancementor inhibition of enzyme activity etc. Assays relying on the ability ofmaterials to alter the behaviour of cells cultured in vitro may also beappropriate, such as assays for cell death, cell proliferation,apoptosis, inhibition or stimulation of cell-to-cell contact, secretionof cytokines or other soluble products, synthesis of specific m-RNA,intracellular vesicular transport, alteration of cell signallingprocesses etc. In vivo assays in whole animals or humans may also becarried out, for example incorporation of radiolabel into thesupramolecular assemblies, followed by investigation of its subsequentdistribution after administration by various routes.

According to this method a combinatorial approach is used in which arange of different supra-molecular assemblies (or “probes”) is prepared,each containing a different combination of conjugates selected from apre-synthesised bank. Selection of the appropriate conjugates may bebased on known properties of the target ligand or may simply involve theuse of a very wide range of head groups to increase the probability thattwo or more of the head groups will form an epitope for the ligand. Inthis way, following the assay for sufficient interaction between theprobe and the ligand as described above, the combination of conjugatesfound to be most effective may be modified by adding further headgroups, removing some head groups, or both, and assaying the resultantprobes once again for sufficient interaction. Eventually, the mostfavourable combination of head groups may be identified and selected foruse in the composition.

The present invention therefore has a very clear advantage overtraditional combinatorial chemistry. In combinatorial chemistry, theidentification of the most favourable sequence for binding to a specificreceptor must be carried out by synthesis of hundreds of possiblecombinations of different groups such as amino acids, in differentorders, each one having to be tested for efficacy. This process istime-consuming, expensive and is limited by the nature of the chemistrywhich can be carried out in linking the different components together.In contrast, the present invention simply relies upon proximity of thehead groups to provide association-derived epitopes. Once a set ofconjugates has been synthesised, no further synthetic chemistry isrequired, only simple mixing of the conjugates to form the differentprobes by non-covalent association.

In a preferred simple embodiment, the present method uses conjugateshaving a single terminal amino acid linked via a spacer to a lipid tailgroup which can be combined simply by mixing in aqueous medium to formmicelles in which different amino acid side chains would be presentedtogether in a multiplicity of different configurations. Accordingly, theneed to present amino acids in a specific order, or with a specificspacing or orientation, is circumvented. On statistical grounds, aproportion of the individual amino acid sub-units will always beassociated in an ideal configuration.

In one arrangement, each of the conjugates would have the linearstructure: X-spacer-spacer-lipid-lipid, where X represents a singleamino acid different for each of the distinct conjugates employed.

When seeking to construct epitopes composed of natural amino acids it ispossible to simplify further the number of head groups for selection.One can categorise the amino acid residues found in naturalproteinaceous materials into six fundamental classes preferably using inany one class one amino acid rather than all members of that classbecause of the increased spatial flexibility of amino acids in theterminal position of the head group. This has the effect of reducingconsiderably the total number of amino acids required for constructingthe pre-synthesised bank of conjugates and thereby the total number ofhead groups used. The main classes of amino acids are set out in Table 1below.

TABLE 1 Class Representative Abbreviation Hydrophobic Leucine LHydroxylic Serine S Acidic Glutamate E Amide Glutamine Q Basic HistidineH Aromatic Tyrosine Y

A number of strategies are available for identifying active combinationsof amino acid-containing conjugates.

In one embodiment, a restricted number of conjugates is employed to forma range of distinct probes where each probe is an aqueous suspension ofsupra-molecular assemblies, each assembly consisting of selectedconjugates mixed together, and each differing from the other as a resultof the inclusion of a different additional conjugate as shown belowwhere each of the letters given represents a conjugate with a differentterminal amino acid:

Probe 1 A B C D Probe 2 A B C E Probe 3 A B C F Probe 3 A B C G . . . .. . Probe x A B C Z

Each of the probes is tested separately in the biological assays forsufficient binding as outlined above.

In a second simple embodiment, an initial probe can be constructed whichcontains a large number of different conjugates from the bank, and itsefficacy compared with probes each lacking a different conjugate inturn, to determine which headgroups in the bank are essential, and whichare redundant for the biological interaction being investigated. Thisapproach is illustrated below:

Probe 1 A B C D E . . . Z Probe 2 A C D E . . . Z Probe 3 A B D E . . .Z . . . Probe x A B C D E . . .

Combinations of the alternative approaches as outlined above can bemade.

A knowledge of the target ligand may assist in designing a suitablestarting array. For example, if the ligand is known to be basic, itwould make sense to impart an acidic character to the conjugates bypresenting them in the form where a free carboxyl group of the terminalamino acid is exposed. Introducing additional functionality by employinga particular amino acid as a spacer group adjacent to the terminal aminoacid may also confer increased specificity. Where the involvement of,say, a short oligo-peptide sequence of known structure has already beenimplicated in binding to the target ligand, such a sequence may beincorporated into a conjugate to be included in the set of conjugatesmaking up the composition.

In a final aspect, the present invention provides a method for producinga molecule for interacting with a ligand. The method comprises producinga composition according to one of the methods defined above; identifyingthe at least two head groups which form an epitope for the ligand in thecomposition; and producing a molecule incorporating the functionalgroups of the at least two head groups optionally spaced apart by one ormore linker groups so that the molecule is capable of interacting withthe ligand more strongly than each of the head groups individually.

Whilst the compositions of the present invention may themselves beuseful in in vitro or in vivo systems perhaps to induce a biologicalresponse in a therapeutic, prophylactic or diagnostic method, in somecircumstances a molecule may be produced based on the structure of theabove compositions. By identifying the functional groups of the at leasttwo head groups which form the epitope for the ligand a new moleculeanalogous to the composition may be produced containing the same or asimilar epitope. The functional groups may, for example, be incorporatedinto a single linear oligo-peptide possibly with one or more linkergroups to space the functional groups apart.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in further detail, by way of exampleonly, with reference to the following Examples and the attacheddrawings, in which:

FIG. 1 shows a schematic representation of the surface of asupra-molecular assembly, and how such a composition according to thepresent invention binds to a target ligand; and

FIG. 2 shows a schematic representation of the surface of asupra-molecular assembly composed of two non-identical conjugates whoseheadgroups consist of short-chain linear peptides.

DETAILED DESCRIPTION OF THE INVENTION

Referring to FIG. 1, a section 1 of a composition according to thepresent invention is shown in the form of a micelle in which the headgroups 2 and tail groups 3 together form conjugates 4 (FIG. 1A). Atarget ligand 5 is presented to the composition 1. Because theconjugates are movable, a rearrangement occurs (FIG. 1B) to allowpositioning of the head groups 2 to bind the target ligand 5. Referringto FIG. 2, a section of a composition according to the present inventionis shown in the form of a supramolecular assembly, in which binding of aligand to the surface of the assembly is brought about by the creationof an epitope constructed via the non-covalent association of twoconjugates composed of short-chain peptides (A), this epitope being ableto interact with the ligand more strongly than either of the individualconjugates in isolation (B). The same principle applies for headgroupscontaining structures other than amino acids.

EXAMPLES

In the examples given below, the standard convention for representationof amino acids by single letters of the alphabet is employed, exceptthat in all cases the letter refers to conjugates as described above inwhich that particular amino acid occupies the terminal position in thepeptide chain. In the examples described here, the lipid comprises twoamino acids linked via a peptide bond, in which both of the amino acidsare glycine analogues, where in each case the alpha hydrogen has beenreplaced by a linear hydrocarbon chain containing either 12 or 14carbons. Linkages between the headgroup and spacer and the spacer andlipid are all via peptide bonds. The headgroup bears a free amino groupand the free end of the lipid bears a CONH₂ group. The structure of eachconjugate is thus: NH₂-headgroup-spacer-amino acid (C₁₄ sidechain)-amino acid (C₁₂ side chain)-CONH₂.

Example 1 Stimulation of TNF Secretion from Macrophages

-   1. Individual conjugates E, Y, Q, S & H (linked to lipid via a    serine-glycine spacer) were prepared as solutions in    methanol/dichloromethane 1:1 at a concentration of 5 mg/ml.-   2. Solutions of the conjugates were dispensed into 7 ml glass vials    in equal proportions, to give a final volume of 400 ul (2 mg of    solid) in all vials, as shown in the example overleaf. In cases    where the volume of organic solution available was insufficient,    adjustment was made at a later stage, when the quantity of water    added for reconstitution was reduced accordingly, as shown.-   3. The contents of all vials were dried down under a stream of    nitrogen, then exposed to a vacuum of at least 1 mbar overnight in a    lyophiliser.-   4. On the following day, distilled water was added in volumes as    indicated in the table overleaf, to give a final concentration in    all vials of 1 mg/ml. The vials were capped, warmed to 37 deg C. and    bath-sonicated until clarity was achieved.-   5. The samples were then applied to wells of 24-well cluster plates    into which cells of the J774A-1 macrophage cell line had been plated    (5×10⁴ cells/ml/well). Volumes of 100 ul and 10 ul of sample were    added to individual wells, and the cells were incubated overnight at    37 deg C. in an atmosphere of 5% CO₂/air.-   6. The following day, duplicate volumes of 50 ul of supernate were    taken from each well and measured for TNF concentration in a capture    ELISA assay. Results obtained are shown in the table below.

Volume of Volume of conjugate dispensed water E Y Q S H added E 260 ul1.3 ml Y 400 ul 2.0 ml Q 310 ul 1.55 ml  S 360 ul 1.8 ml H 400 2.0 ml EY200 ul 200 ul 2.0 ml EQ 200 ul 200 ul 2.0 ml ES 200 ul 200 ul 2.0 ml EH200 ul 200 ul 2.0 ml YQ 200 ul 200 ul 2.0 ml YS 200 ul 200 ul 2.0 ml YH200 ul 200 ul 2.0 ml QS 200 ul 200 ul 2.0 ml QH 200 ul 200 ul 2.0 ml SH200 ul 200 ul 2.0 ml QSH 133 ul 133 ul 133 ul 2.0 ml YSH 133 ul 133 ul133 ul 2.0 ml YQH 133 ul 133 ul 133 ul 2.0 ml YQS 133 ul 133 ul 133 ul2.0 ml ESH 133 ul 133 ul 133 ul 2.0 ml EQH 133 ul 133 ul 133 ul 2.0 mlEYH 133 ul 133 ul 133 ul 2.0 ml EYS 133 ul 133 ul 133 ul 2.0 ml EYQ 133ul 133 ul 133 ul 2.0 ml EQS 133 ul 133 ul 133 ul 2.0 ml EYQS 50 ul 50 ul50 ul 50 ul 1.0 ml EYQH 50 ul 50 ul 50 ul 50 ul 1.0 ml EYSH 50 ul 50 ul50 ul 50 ul 1.0 ml EQSH 50 ul 50 ul 50 ul 50 ul 1.0 ml YQSH 50 ul 50 ul50 ul 50 ul 1.0 ml EYQSH 40 ul 40 ul 40 ul 40 ul 40 ul 1.0 ml

OD₄₅₀ in J774 supernates 100 ug 10 ug 0 ug E 0.628 0.098 0.013 Y 0.3130.053 Q 0.083 0.015 S 0.348 0.143 H 0.632 0.206 EY 0.198 0.027 EQ 0.1130.022 ES 0.211 0.225 EH 0.167 0.037 YQ 0.245 0.034 YS 0.786 0.363 YH0.541 0.133 QS 0.212 0.025 QH 0.135 0.027 SH 0.515 0.177 QSH 0.253 0.032YSH 0.712 0.229 YQH 0.290 0.020 YQS 0.519 0.119 ESH 0.380 0.246 EQH0.107 0.026 EYH 0.254 0.042 EYS 1.289 0.355 EYQ 0.191 0.064 EQS 0.2090.027 EYQS 0.777 0.206 EYQH 0.224 0.067 EYSH 0.262 0.146 EQSH 0.1490.185 YQSH 0.319 0.045 EYQSH 0.375 0.073

It can be seen that some, but not all, of the combinations of differentheadgroups elicit strong biological responses, indicating that theresponse is specific to those particular combinations. The exampleillustrates the way in which the conjugates described can be employed inthe combinatorial approach to identify efficacious combinations for thepurpose of eliciting a desired biological response.

Example 2 TNF Secretion from Macrophages

Comparison of Supra-molecular Assemblies Containing a Mixture ofConjugates, with a Mixture of Supra-molecular Assemblies Each Containinga Single Conjugate

Samples were prepared as described in Example b 1, with or without theinclusion of additional lipidic materials as described below. Thecombination of conjugates Y, S and L was chosen since this combinationwas a good performer in the experiment described in Example 1.

Probes containing phosphatidyl choline were prepared at a ratio ofphospholipid to conjugate of 2:1 wt/wt.

Probes containing octyl glucoside were prepared at a ratio of glycolipidto conjugate of 1:1 wt/wt.

Results shown in the table below are optical densities at 450 nm of TNFELISAs conducted on 18 hour culture supernatants. The concentration ofconjugate in the wells was 10 μug/ml

OD₄₅₀ of TNF ELISA EYS 0.390 E + Y + S 0.059 medium control 0.000 EYS:OG0.559 (E + Y + S):OG 0.193 OG control 0.228 EYS:PC 0.320 (E + Y + S):PC0.130 PC control 0.081

This example shows that combinations of the conjugates can elicitbiological responses either when presented alone, or when presented inconjunction with other lipids, such as phospholipids or lipid sugars. Italso shows that for efficacy to be manifested, it is important for allof the conjugates to be presented in combination on the samesupra-molecular assembly, and that activity is not observed if the sameconjugates are presented together at the same time, but separated ondifferent supra-molecular assemblies. This suggests that it is importantto present the conjugates in close proximity to each other, in order topermit the formation of epitopes formed by non-covalent association ofthe conjugates, which can participate in specific binding withcell-surface receptors.

Example 3 Enhancement of Oral Uptake

-   1. Individual conjugates L, S, E & Q (conjugated to lipid via a    tyrosine-glycine spacer) were prepared as solutions in benzyl    alcohol at a concentration of 10 mg/ml.-   2. 75 ul of ¹⁴C-cholesterol oleate (3.7 MBq/ml in toluene) was    dispensed into four 7 ml glass screw-capped vials and dried down    under a stream of nitrogen.-   3. 400 ul of each of the solutions in (1) was added to one of the    vials in (2) and shaken overnight at room temperature.-   4. Solutions of the conjugates were dispensed into 7 ml glass vials    in equal proportions, to give a final volume of 80 ul (0.8 mg of    solid) in all vials, as shown in the example below.

L S E Q L 80 ul — — — S — 80 ul — — E — — 80 ul — Q — — — 80 ul LS 40 ul40 ul — — LE 40 ul — 40 ul — LQ 40 ul — — 40 ul SE — 40 ul 40 ul — SQ —40 ul — 40 ul EQ — — 40 ul 40 ul LSE 27 ul 27 ul 27 ul — LSQ 27 ul 27 ul— 27 ul LEQ 27 ul — 27 ul 27 ul SEQ — 27 ul 27 ul 27 ul LSEQ 20 ul 20 ul20 ul 20 ul

-   5. 2 ml of distilled water was added to each of the vials with    vortexing. The vials were then capped and bath-sonicated for 20    minutes.-   6. The samples were then frozen in liquid nitrogen and lyophilised    overnight.-   7. The following day, each vial was reconstituted with 2 ml of    distilled water and sonicated again until clear dispersions were    achieved.-   8. The samples were administered by oral gavage to Balb/c female    mice (20-25 g weight—four mice per group) at a dose of 0.3 ml per    animal.-   9. 75 ul heparinised blood samples were taken by tail venupuncture    at 45, 90 and 180 minutes after administration.-   10. Each sample was diluted in 0.5 ml of PBS, which was then    centrifuged, and 0.4 ml of the supernate was transferred to a    scintillation vial to which 2 ml of Optiphase Hisafe 3 (Wallac) was    added with mixing.-   11. Activity in the samples was measured in a scintillation counter.

Percentage uptake was estimated on the basis of a 2 ml blood volume, ofwhich 1 ml was assumed to be plasma.

Results are shown in the table below.

% uptake in bloodstream 45 mins 90 mins 180 mins L 0.90 1.39 0.61 S 1.121.14 0.81 E 0.85 1.55 0.79 Q 1.40 3.00 0.81 LS 2.87 2.38 0.66 LE 2.592.22 0.49 LQ 5.05 2.15 0.45 SE 4.21 1.66 0.70 SQ 4.67 1.45 0.67 EQ 3.722.65 0.59 LSE 1.91 1.20 0.97 LSQ 6.23 1.90 0.80 LEQ 2.77 1.73 0.98 SEQ3.06 1.52 0.63 LSEQ 2.45 1.74 0.81

It can be seen that some, but not all, of the combinations of differentheadgroups enhance uptake of label via the oral route, indicating thatthe response is specific to those particular combinations. The exampleillustrates the way in which the conjugates described can be employed inthe combinatorial approach to identify efficacious combinations capableof acting as targeting ligands.

Example 4 ELISA Fc Binding

-   1. 100 ul of goat IgG (1 mg/ml) was added to 20 ml of PBS and 100 ul    was placed in each well of a flat-bottomed microtitre plate.-   2. The plate was incubated for several days at +4 deg C.-   3. 2 mg of each of the conjugates Y, F, W, L, S, E, Q & R (each    linked to lipid via a serine-glycine spacer) were weighed into 1 ml    glass vials and 200 ul of benzyl alcohol added to give solutions of    each conjugate at a concentration of 10 mg/ml.-   4. The solutions were dispensed in 7 ml glass screw-capped vials as    follows:

Vial No. Y F W L S E Q R 1 20 ul 20 ul 20 ul — 2 20 ul 20 ul — 20 ul 320 ul — 20 ul 20 ul 4 — 20 ul 20 ul 20 ul 5 20 ul 20 ul 20 ul — 6 20 ul20 ul — 20 ul 7 20 ul — 20 ul 20 ul

-   5. The contents of each vial were mixed well by vortexing, then 1.5    ml of distilled water was added to each vial.-   6. The vials were capped and bath-sonicated for five minutes to give    crystal clear dispersions.-   7. The plate from step (2) was washed in PBS/0.02% Tween 20 and then    blocked by incubating for one hour with 1% BSA in PBS (300 ul/well).-   8. The plate was then washed as before, and 100 ul of sample from    each of the vials in step (6) was added to wells in column (1) of    rows (1) to (7) Row (8) was left as a blank control.-   9. Doubling dilutions were performed across the plate by    transferring 100 ul from wells in column (1) to the adjacent well on    the same row in column (2) and mixing, then transferring 100 ul to    the next column as before, etc.-   10. The plate was then incubated overnight at +4 deg C.-   11. The following day, the plate was washed as before and 10 ul of    commercial horseradish peroxidase-IgG conjugate (diluted 1/1000 in    PBS) was added to each well and incubated at room temperature for 40    minutes.-   12. The plate was then washed again, and 100 ul of OPD substrate for    peroxidase was added to each well and incubated at room temperature    for 30 minutes.-   13. 20 ul of 3M sulphuric acid was then added to each well to stop    the reaction.-   14. The optical density of each of the wells was measured at 450 nm    on a plate reader, and the results obtained, after adjustment for    background, are recorded below.

Sample 1 in 4 1 in 8 1 in 16 1 in 32 1 in 64 1 YFW 0.001 0.039 0.0480.053 0.083 2 YFL 1.504 1.484 1.325 0.723 0.051 3 YWL 0.803 0.192 0.0220.023 0.060 4 FWL 1.034 0.778 0.208 0.031 0.034 5 SEQ 0.029 0.041 0.0550.057 0.091 6 SER 0.013 0.030 0.044 0.062 0.075 7 SQR 0.000 0.045 0.0310.054 0.065

It can be seen that maximal binding is achieved with samples 2, 3 and 4(ie combinations YFL, YWL, and FWL).

It can be seen that some, but not all, of the combinations of differentheadgroups enter into strong binding interactions, indicating that theresponse is specific to those particular combinations. The exampleillustrates the way in which the conjugates described can be employed inthe combinatorial approach to identify efficacious combinations for thepurpose of eliciting a desired binding interaction.

1. A method for producing a composition for interacting with a ligand,which method comprises: (a) providing a plurality of distinctconjugates, each conjugate comprising a head group and a tail group; and(b) forming from the plurality of conjugates a non-covalent associationthereof, in which the tail groups aggregate hydrophobically and in whichthe conjugates are movable so that, in the presence of a ligand, atleast two of the head groups are appropriately positioned to form anepitope capable of interacting with the ligand more strongly than eachof head groups individually.
 2. The method of claim 1, wherein the headgroup is selected from the group consisting of an amino acid, a peptide,a peptide analogue, a monosaccharide, a polysaccharide, amononucleotide, a polynucleotide, a sterol, a water-soluble vitamin, aporphyrin nucleus, a haem nucleus, a metal ion chelate, a water-solubledrug, a hormone, and an enzyme substrate.
 3. The method of claim 2,wherein the head group comprises an amino acid.
 4. The method of claim3, wherein the head group comprises a peptide.
 5. The method of claim 3or claim 4, wherein the amino acid comprises a terminal amino acidselected from the group consisting of hydrophilic amino acids,hydroxylic amino acids, acidic amino acids, amide amino acids, basicamino acids, and aromatic amino acids.
 6. The method of claim 1, whereinthe tail group comprises a lipophilic group selected from the groupconsisting of a straight chain fatty acid, a branched-chain fatty acid,an alcohol having at least 8 carbon atoms, an aldehyde having at least 8carbon atoms, a lipidic amino acid analogue, a prostaglandin, aleukotriene, a monoglyceride, a diglyceride, a sterol, a sphingosinederivative, a ceramide derivative, a silicon-substituted derivativethereof, and a halogen-substituted derivative thereof.
 7. The method ofclaim 6, wherein the lipophilic group comprises a C₁₀ to C₁₄ fatty acid.8. The method of claim 1, wherein the conjugate further comprises aspacer group linking the head group to the tail group.
 9. The method ofclaim 8, wherein the spacer group is hydrophilic.
 10. The method ofclaim 9, wherein the spacer group comprises an amino acid, a hydroxyacid, a sugar or a polyethylene glycol.
 11. The method of claim 1,wherein the non-covalent association comprises a lamellar structure, amicelle or a liposome.
 12. The method of claim 1, wherein the step ofproviding the plurality of conjugates comprises: (a) selecting a set ofconjugates with an array of head groups; (b) forming a non-covalentassociation therefrom, in which the tail groups aggregatehydrophobically and in which the conjugates are movable; (c) assayingfor sufficient interaction between the non-covalent association and theligand; (d) optionally repeating steps (a) to (c) using a set ofconjugates with a modified array of head groups; and (e) on findingsufficient interaction in step (c) selecting the set of conjugates asthe plurality of conjugates in step (a).
 13. The method of claim 12,wherein the array of head groups comprises (a) at least one terminalamino acid from each of the following classes of amino acid: hydrophobicamino acids, hydroxylic amino acids, acidic amino acids and amide aminoacids; and (b) at least two further terminal amino acids comprising atleast one basic amino acid and at least one aromatic amino acid, or atleast two basic amino acids or aromatic amino acids.
 14. The method ofclaim 13, wherein the modified array of head groups used in step (d)comprises the array of head groups used in steps (a) to (c) in which theat least two further terminal amino acids are different from those usedin steps (a) to (c).
 15. The method of claim 12, wherein the array ofhead groups comprises at least one terminal amino acid from each of thefollowing classes of amino acid: hydrophobic amino acids, hydroxylicamino acids, acidic amino acids, amide amino acids, basic amino acidsand aromatic amino acids.
 16. The method of claim 15, wherein themodified array of head groups used in step (d) comprises the array ofhead groups used in steps (a) to (c) in which the at least one terminalamino acid from one of the classes of amino acid is either absent orreplaced by a charged version thereof.
 17. A method for producing amolecule for interacting with a ligand, comprising: (a) producing acomposition according to the method of claim 1; (b) identifying the atleast two head group which form an epitope for the ligand in thecomposition; and (c) producing a molecule incorporating the functionalgroups of the at least two head groups optionally spaced apart by one ormore linker groups so that the molecule is capable of interacting withthe ligand more strongly than each of the head groups individually. 18.A method of treating a disease comprising administering to a patientisolated micelles which comprise a plurality of conjugate moleculesnon-covalently associating with one another to form the micelles, eachconjugate molecule comprising (1) a head group molecule conjugated to ahydrophobic tail group molecule, optionally via a spacer molecule, (2) asurface formed by the head group molecules, which surface comprises aplurality of distinct non-covalent associations of the head groupmolecules, and (3) a hydrophobic core formed by the hydrophobic tailgroup molecules; wherein the head group molecules in the non-covalentassociations change configuration through the movement of the head groupmolecules on or along the surface, and the movement of the head groupmolecules is facilitated by the movement of the conjugate molecules inthe micelle, and wherein a distinct non-covalent association of the headgroup molecules forms an epitope which has higher affinity to a ligandthan each of the head groups of the conjugates individually does. 19.The method of claim 18 wherein the head group of the micelles isselected from the group consisting of an amino acid, a peptide, apeptide analogue, a monosaccharide, a polysaccharide, a mononucleotide,a polynucleotide, a sterol, a water-soluble vitamin, a porphyrinnucleus, a haem nucleus, a metal ion chelate, a water-soluble drug, ahormone, and an enzyme substrate.
 20. The method of claim 19 wherein thehead group comprises an amino acid.
 21. The method of claim 20, whereinthe head group comprises a peptide.
 22. The method of claim 20 or ofclaim 21, wherein the amino acid comprises a terminal amino acidselected from the group consisting of hydrophilic amino acids,hydroxylic amino acids, acidic amino acids, amide amino acids, basicamino acids, and aromatic amino acids.
 23. The method of claim 18,wherein the tail group comprises a lipophilic group selected from thegroup consisting of a straight chain fatty acid, a branched-chain fattyacid, an alcohol having at least 8 carbon atoms, an aldehyde having atleast 8 carbon atoms, a lipidic amino acid analogue, a prostaglandin, aleukotriene, a monoglyceride, a diglyceride, a sterol, a sphingosinederivative, a ceramide derivative, a silicon-substituted derivativethereof, and a halogen-substituted derivative thereof.
 24. The method ofclaim 23, wherein the lipophilic group comprises a C₁₀ to C₁₄ fattyacid.
 25. The method according to claim 18, wherein the conjugatefurther comprises a spacer group linking the head group to the tailgroup.
 26. The method according to claim 25, wherein the spacer group ishydrophilic.
 27. The method according to claim 26, wherein the spacergroup comprises an amino acid, a hydroxy acid, a sugar or a polyethyleneglycol.